Semi-synthetic sulphaminoheparosansulphates having high anti-metastatic activity and reduced haemorrhagic risk

ABSTRACT

Sulphaminoheparosansulphates obtainable from the  Escherichia coli  K5 polysaccharide by deacetilation and the subsequent sulfation with the sulphuric anhydride/trimethylamine adduct carried out at 0° C., for times ranging from 0.25 to 2 hours and using a reactant/polysaccharide ratio (SO 3  equivalents/available OH groups equivalents) equal to 5 have been found having high anti-metastatic activity and low anticoagulant activity.

This application is a U.S. National Stage entry under 35 U.S.C. §371 ofPCT/EP97/04682, filed Aug. 28, 1997.

The object of the present invention is the use of thesulphaminoheparosansulphates as anti-metastatic agents.

The metastasis is a process consisting of the detachment of cancer cellsfrom the site of the primary cancer, the dissemination in the bloodflow, the adhesion to the vascular walls, and the migration and growthin extra-vascular spaces. Said phenomena, and in particular the adhesionto the vascular walls, seem to be regulated by the endogenous heparansulfate (HS) polysaccharide. Some anticoagulant drugs, among which theheparin (HEP), which shows structural analogies with the heparansulfate, have been tested as potential anti-metastatic agents. (I.Vlodavsky et al.: “Modulation of neovascularization and metastasis byspecies of heparin”, in: “Heparin and Related Polysaccharides” (D. A.Lane et al., Eds.), Plenum Press, New York 1992, 317-327). The heparinamong said drugs is particularly active as anti-metastatic, but its highanticoagulant activity implies haemorrhagic risks, whereby the searchfor heparin-like substances having reduced anticoagulant activity isparticularly interesting. (D. J. Tyrrell et al.: “Therapeutic uses ofheparin beyond its traditional role as an anticoagulant”, TIPS 16,198-204, 1995).

With the present invention we have found that some semi-syntheticheparan sulfates belonging to the sulphaminoheparosansulphates (SAHS)class (B. Casu et al.,: “Heparin-like compounds prepared by chemicalmodification of capsular polysaccharide from E. coli K5”, Carbohydr.Res. 263, 271-284 (1994)), surprisingly carry on an “in vivo”anti-metastatic activity comparable to the heparin one, even if havingan “in vivo” anticoagulant activity an order of magnitude lower than theheparin one.

More particularly, we have found that only the SAHS obtainable from theEscherichia coli K5 polysaccharide by deacetilation and subsequentsulfation with the sulphuric anhydride/trimethylamine adduct carried outat 0° C., for times ranging from 0.25 to 2 hours and using areactant/polysaccharide ratio (SO₃ equivalents/available OH groupsequivalents) equal to 5 (named SAHS-B), having molecular weight rangingfrom 5,000 to 40,000, show anti-metastatic activity comparable to atypical heparin one, while SAHS prepared according to other experimentalconditions have anti-metastatic activity notably lower either than theheparin one or than the SAHS-B one.

Moreover we have also found that fractions of SAHS-B having a molecularweight lower than 5,000 keep a significant anti-metastatic activity(also greater than the one of the corresponding heparins having lowmolecular weight).

Therefore the semi-synthetic SAHS-B heparosansulfates look asanti-metastatic drugs having a reduced haemorrhagic risk.

For the purpose, the SAHS-B will be formulated in suitablepharmaceutical compositions, using conventional techniques andexcipients. Such compositions may be administered for the prevention orthe therapy of metastases in doses which will obviously depend fromseveral factors but which will be generally ranging from 1 to 1,000 mgof SAHS-B one or more times a day.

The SAHS have been obtained as previously described (B. Casu et al.,1994, loc. cit.; PCT/EP94/01660) from the K5 polysaccharide, which is aconstituent of the cell membrane of the Escherichia coli K5 strain. Inparticular, the K5 polysaccharide has been selectively N-deacetilatedand N-sulfated, and then O-sulfated as summarily described in thefollowing scheme, obtaining the SAHS of different kind SAHS-B, SAHS-C,SAHS-A.

Scheme

The first step consists of the N-deacetilation by hydrazinelysis of theK5 polysaccharide (K5-PS). The obtained product (heparosan, AH) isN-sulfated with the sulphuric anhydride/trimethylamine adduct (TMA/SO₃),with the achievement of the sulphaminoheparosan (SAH). The numbers nearthe arrows show in order the reaction temperatures (° C.), thereactant/polysaccharide ratios (SO₃ equivalents/equivalents of availablehydroxyl groups), and the reaction times (hours).

The anti-metastatic activity of the SAHS, heparin and other referencesulfated polysaccharides has been tested using the method of thecolonization to the lung of B16B16 melanoma cells. (N. Caselia et al.,Thromb. Haem. 73,964 (1995)). Such a method, lending itself particularlyto test the effect of the drugs with inhibitory activity on the cancerhaematic dissemination, consists of the evaluation of the number of thecancer colonies which form in the lung after the injection of murinemelanoma cells by intravenous way in the mouse. B16B16 melanoma cellshave been used. The cells have been cultured in DME with 10% of fetalbovine serum in a CO₂ (5%) incubator in humidity conditions and at 37°C. The cells have been divided two times a week, treating them with0.25% trypsin/0.05% EDTA. The polysaccharides to test have beendissolved in physiological solution or in phosphate (PBS) buffer, at theproper dilution, and used on the spot. B16B16 melanoma cells, diluted inPBS (10⁵ cells/0.1 ml/mouse) have been injected into a side vein of thetail of C57B16 mice having an average weight equal to 20 g, in a finalvolume equal to 0.2 ml/mouse. The mice have been sacrificed 12-16 daysafter the injection of the cancer cells; the lungs have been taken andfixed in a Buoin solution for the count of the superficial metastaticnodules, which are pointed out as black masses on a yellow ground. Thenthe ratio between the number of lung nodules in the treated mice and inthe control ones has been estimated. Each experiment has been carriedout on a minimum number of five mice, more frequently on 8-10 mice. Theinhibition percentages of the metastases discovered in severalexperiments have been reported in the individual Examples and in Table1.

EXAMPLE 1

Preparation and anti-metastatic activity of type A, B and C SAHS

Standard procedures for the preparation of somesulphaminoheparosansulphates having different anti-metastatic activityare hereinafter described. The products have been characterized withrespect to the average molecular weight (by gel filtration), sulfationdegree (expressed as sulfates/carboxyles molar ratio, determined byconductimetry), and distribution of the sulfate groups (determined by ¹Hand ¹³C NMR spectrometry), as described in Casu et al., 1994 (loc.cit.). The procedures described hereinbelow take to SAHS having aN-sulfation degree about equal to 100%, and a 6-O-sulfation degree atleast equal to 25%.

The starting K5 polysaccharide may be suitably prepared as described inthe Italian Patent Application M191A000659.

The quantities in brackets are indicative.

1a) N-deacetilation

The K5 polysaccharide (100 mg) and hydrazine sulfate (138 mg) aredissolved in anhydrous hydrazine (1.38 mg) and maintained in a closedpipe, under nitrogen atmosphere, for 5 hours at 96° C. The solution isdried in a rotating evaporator, the reaction product is dissolved indistilled water and the pH is taken to 4 with 37% HCl. The pH is takento 9 with NaOH 2N and 4 volumes of ethanol saturated with sodium acetateare added. The obtained precipitate is filtered, dissolved in distilledwater, and the solution is dialyzed against distilled water for 3 days(3×2l each day; cut-off 14,000 D) and finally freeze-dried.

1b) N-sulfation

The polysaccharide obtained as in 1a) (100 mg) is dissolved in distilledwater, the solution pH is taken to 9 by the addition of solid sodiumbicarbonate, and the temperature increased to 55° C. At thistemperature, maintaining the mixture under stirring, 100 mg oftrimethylamine/sulfur trioxide adduct (TMA/SO₃) are added. Equal amountsof the adduct are added after 4 hours, and it is left to react for atotal time equal to 24 hours. The recovery of the N-sulfatedpolysaccharide is carried out as described above.

1c) N-sulfation

The polysaccharide obtained as in 1b) (100 mg) is dissolved in distilledwater (20 ml), and the solution is passed through an Amberlite IR-120H⁺column at room temperature. The column is washed with other 20 ml ofdistilled water and the eluates are collected, which are taken to pH 5.5with 10% tributylamine in ethanol (w/v) (3 ml). The tributylamine excessis removed with diethyl ether (40 ml) and it is freeze-dried.

The so obtained product (188.2 mg) is dissolved in anhydrousdimethylformamide (33 ml), the pyridine/sulfur trioxide adduct (Py/SO₃,amounts indicated below) dissolved in 15 ml of anhydrousdimethylformamide is added, and the reaction mixture is maintained atthe temperatures and for the times indicated below. In order to obtaindifferent types of SAHS, different reaction temperatures, amounts ofsulfur adduct and reaction times have been adopted. In particular, thetype A SAHS has been obtained working at 0° C., and using 460 mg ofpyridine/SO₃, for 1 hour. The product (G1524-3; average molecular weight11,700; sulfates/carboxyles molar ratio 1.8) has shown ananti-metastatic activity corresponding to 17.5% of metastasis inhibitionfor a dose equal to 0.5 mg/mouse; in the same test, the referenceheparin has shown the 97.5% of inhibition, and 54.8% for the heparansulfate from pig-pancreas.

The type B SAHS has been obtained working at 0° C., using 765 mg ofsulfur adduct, for 0.25-2 hours (preferably 1 hour) and submitting againthe product to N-resulfation as described in 1b). A typical finalproduct (G1669; average molecular weight 25,700; sulfates/carboxylesmolar ratio 2.2) has shown an anti-metastatic activity (0.5 mg/mousedose) corresponding to 92.7% of metastasis inhibition.

The type C SAHS has been obtained working at 25° C. for 1 hour, with7.650 mg of sulfur adduct. The product (G1524/3; average molecularweight 10,800; sulfates/carboxyles molar ratio 2.8) has shown ananti-metastatic activity (0.5 mg/mouse dose) corresponding to 8.8% ofmetastasis inhibition.

EXAMPLE 2

Type B SAHS anti-metastatic activity

The anti-metastatic activity tests have been repeated for the SAHS-Bprepared as described in the Example 1 (product G1669), for three doses(0.5; 0.2 and 0.1 mg/mouse). The corresponding inhibitions of themetastases have been respectively 78.5%, 62.5% and 20.5%; for the samedoses, the reference heparin has shown inhibitions respectively equal to95.5%, 91.3% and 80.3%.

EXAMPLE 3

Type B SAHS anti-metastatic activity

The anti-metastatic activity test has been repeated for the type B SAHSprepared as described in the Example 1 (product G1669), for the dose 0.5mg/mouse, showing an inhibition equal to 98.5% of the metastases. (Atthe same dose, the reference heparin has shown an inhibition equal to98.5%, and for a “super-sulfated” heparin having a low molecular weightan inhibition equal to 91.0%.

EXAMPLE 4

Preparation and anti-metastatic activity of type B SAHS fractions havingdifferent molecular weight

A sample of type B SAHS (preparation G1668, obtained essentially asdescribed in the Example 1) has been fractionated by Sephadex gelchromatography, and the three fractions characterized by analogoussulfates/carboxyles (2.2-2.3) ratios and different molecular weightshave been isolated: G1668a (average molecular weight 38,200), G1668c1(22,700) and G1668b1 (3,200). The corresponding anti-metastaticactivities (0.5 mg/mouse dose) turned out to be analogous for the threefractions (inhibition equal to 97-98%) and analogous to another nonfractionated SAHS-B preparation (G1783) one prepared as described in theExample 1. In the same set of experiments, the reference heparin hasshown an inhibition equal to 95-97%.

EXAMPLE 5

Anti-metastatic activity of low molecular weight SAHS-B

Comparison with other natural and super-sulfated glycosaminoglycans. Theanti-metastatic activity of the G1668b1 fraction having low molecularweight (obtained as described in the Example 4) turned out to correspondto 83.6% of metastasis inhibition. In the same test, the referenceheparin has shown an inhibition equal to 92.8%, a “super-sulfated”heparan sulfate having low molecular weight (ssLMWHS) an inhibitionequal to 46.4%, and a dermatan sulfate 4.6-disulfated (DS4, 6S) aninhibition equal to 65.32%.

EXAMPLE 6

SAHS-B anti-metastatic activity

A preparation of SAHS-B (product G1668, obtained acting essentially asdescribed in the Example 1) has shown an anti-metastatic activity (0.5mg/mouse dose) corresponding to the 98.5% of metastasis inhibition. (Inthe same test, the reference heparin has given 98.5% of inhibition).

EXAMPLE 7

Anti-metastatic activity of a low dose of a low molecular weight SAHS-Bfraction

The low molecular weight G1668c1 fraction described in the Example 4 hasshown, at the dose equal to 0.1 mg/mouse, an anti-metastatic activitycorresponding to the inhibition of the 41.0% of the metastases. (At thesame dose, the non fractionated reference heparin provided an inhibitionequal to 91.1%).

EXAMPLE 8

Anti-metastatic activity of a very low dose of a low molecular weightSAHS-B fraction

The low molecular weight G1668c1 fraction described in the Example 4 hasshown, at the dose equal to 0.02 mg/mouse, an anti-metastatic activitycorresponding to the inhibition of the 24% of the metastases. (At thesame dose, the reference heparin inhibited 30% of the metastases).

EXAMPLE 9

Anticoagulant activity of SAHS-B

The G1668 product anticoagulant activity, determined as the prolongationof the APTT value in the mouse (intravenous injection of 0.5 mg/mouse;experiments on a group of 4 mice), turned out to be respectively >300;44.4, and 39.9 respectively after 1, 2, and 4 hours from the injection.The corresponding values for the reference heparin havebeen: >300; >300, and 37.6. (Common value for the controls: 28.5).

TABLE 1 ANTI-METASTATIC ACTIVITY OF THE SULFAMINO-HEPAROSANSULFATES(SAHS) dose (mg/mouse) inhibition % N.1 SAHS-A (G1524-3) 0.5 17.5 (HEP97.5; HS 54.8) SAHS-B (G1669) 0.5 92.7 SAHS-C (G1655NS) 0.5  8.8 N.2SAHS-B (G1669) 0.5 75.8 (HEP 95.5) 0.2 62.5 (91.3) 0.1 20.5 (80.3) N.3SAHS-B (G1669) 0.5 84.7 (HEP 98.5; ssLMW-LMW 91.0) N.4 LMW-SAHS-B(G1668c1) 0.5 97-98 (HEP 95-97; LMW-HEP˜50) SAHS-B (G1668b1) 0.5 97-98SAHS-B (G1668a) 0.5 97-98 SAHS-B (G1783) 0.5 97-98 N.5 SAHS-B (G1668b1)0.5 86.3 (HEP 92.8; ssLMW-HS 46.4; DS4, 6S 65.3) N.6 SAHS-B (G1668) 0.597.4 (HEP 98.5) N.7 LMW-SAHS-B (G1668c1) 0.1 41.0 (HEP 91.1) N.8LMW-SAHS-B (G1668c1)  0.02 24 (HEP 30.0).

What is claimed is:
 1. A process for the preparation of pharmaceuticalcompositions having antimetastatic activity and a reduced hemorrhagicrisk, containing a sulfaminoheparosan sulfate, comprising: a)deacetylation of K5 polysaccharide from Escherichia coli to obtaindeacetylated K5 polysaccharide; b) sulfation of deacetylated K5polysaccharide obtained in step a) with sulfuricanhydride/trimethylamine adduct to obtain sulfaminoheparosan sulfate,and c) formulating said sulfaminoheparosan sulfate with conventionalexcipients, wherein said sulfation is carried out at 0° C. for timesranging from 0.25 to 2 hours and using an adduct/polysaccharide ratio,defined as SO₃ equivalents/available OH groups equivalents, equal to 5.2. A process according to claim 1, wherein said sulfaminoheparosansulfate has an average molecular weight ranging from 3,200 to 38,200 D.3. A process according to claim 1, wherein said sulfaminoheparosansulfate has a sulfates/carboxyls ratio of 2.2-2.3.
 4. A therapeuticmethod for therapy of metastases consisting in the administration topatients in need of therapy for metastases of a composition comprising asulfaminoheparosan sulfate prepared according to claim 1, in an amountcorresponding to from 1 to 1,000 mg of said sulfaminoheparosan sulfateone or more times a day.